Our central goal is to understand the molecular basis for the chemical regulation of gap junctions and its[unreadable] implications on cell behavior. This project has been a part of the Program Project Grant since its inception in[unreadable] 1990. Recently, we have shown direct, pH-dependent intramolecular association between the carboxyl[unreadable] terminal (CT) domain and the second half of the cytoplasmic loop of Cx43. We propose that specific peptides[unreadable] can be designed to interfere with this interaction and modulate Cx43 regulation. We have used a highthroughput[unreadable] phage display assay to identify peptides that bind to Cx43CT. We found that Cx43CT[unreadable] preferentially bound to peptides containing a specific "RXP" motif. Several RXP peptides bind to Cx43CT[unreadable] with micromolar affinity, cause structural modifications in the Cx43CT domain and modify Cx43 channel[unreadable] function and regulation. It is the goal of this proposal to further characterize these peptides and search for[unreadable] analogous sequences which may modulate the function of Cx43 channels. The specific aims are: Aim 1.) To[unreadable] characterize the high-order structure of RXP/Cx43CT complexes. Aim 2.) To characterize the effects of RXP-[unreadable] 1 and RXP-4 on the regulation of Cx43 channels. Aim 3.) To identify the structural constraints mediating the[unreadable] binding of RXP peptides to Cx43CT and their functional effects on Cx43 channels. We will use an iterative[unreadable] combination of spectroscopic methods (Nuclear Magnetic Resonance, Surface Plasmon sonance),[unreadable] proteomics approaches (high-throughput biased phage display; peptide synthesis) and functional assays to[unreadable] develop a structural-activity correlate toward peptides with higher affinity and selective functional effects.[unreadable] These peptides will potentially be probes for the manipulation of Cx43 channels and their regulation. These[unreadable] studies will lead to a better understanding of the role of connexins in health and disease.